【摘要】：[研究背景及目的] 各種類型的頜骨缺損在臨床上十分常見,而傳統(tǒng)的修復(fù)方法如牽引成骨、自體骨移植、異體骨移植等常受到供體數(shù)量不足、供體病變、二次損傷、潛在抗原性、感染等因素的制約而亟待改善。以至于頜骨缺損的修復(fù)問題一直是臨床上難以解決的問題。組織工程學(xué)的發(fā)展為骨缺損修復(fù)帶來了希望,其目地是將功能細(xì)胞聯(lián)合培養(yǎng),與可降解三維生物支架材料構(gòu)建成為有活性的組織或器官,然后植入體內(nèi),替代病損的組織,恢復(fù)其形態(tài)、結(jié)構(gòu)和功能。這種方法特別適合修復(fù)頜骨缺損。但目前組織工程骨構(gòu)建方法雖然能夠成骨,但存在組織工程骨血管化緩慢,新骨生長遲緩生長不穩(wěn)定等問題。 人骨髓間充質(zhì)干細(xì)胞(bone marrow Mesenchymal stem cells, hBMSCs)且有多向分化的潛能,在適當(dāng)條件下不僅可以分化為脂肪細(xì)胞、骨細(xì)胞、軟骨細(xì)胞、心肌細(xì)胞、神經(jīng)元細(xì)胞、成肌細(xì)胞、肌腱細(xì)胞及星形膠質(zhì)細(xì)胞等。目前關(guān)于誘導(dǎo)單純hBMSCs向成骨細(xì)胞分化的研究取得了較大的進(jìn)展,其中以BMP2、BMP4、 BMP6及BMP7的功能最具代表性,能引起多種細(xì)胞增殖、分化、凋亡,參與組織再生和再建(remodeling),在細(xì)胞游走和分裂等多種生命活動(dòng)中發(fā)揮調(diào)節(jié)作用。然而誘導(dǎo)單純hBMSCs向成骨細(xì)胞分化存在成骨周期長、成骨效率低、細(xì)胞易老化等缺點(diǎn)。 內(nèi)皮細(xì)胞可以分泌骨形態(tài)發(fā)生蛋白(Bone morphogenetic protein, BMP),促進(jìn)成骨分化的同時(shí)刺激成骨細(xì)胞及其前體細(xì)胞分泌血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF),而VEGF在血管發(fā)生和形成過程中發(fā)揮著重要作用,可以促進(jìn)內(nèi)皮細(xì)胞增殖。然而目前血管內(nèi)皮細(xì)胞對骨髓問充質(zhì)干細(xì)胞成骨分化作用的具體機(jī)制還不清楚,尚缺乏從基因水平驗(yàn)證血管內(nèi)皮細(xì)胞對骨髓間充質(zhì)干細(xì)胞成骨分化的作用。Smad蛋白家族是近年新發(fā)現(xiàn)的細(xì)胞內(nèi)信號傳導(dǎo)蛋白,直接參與TGF-B超家族成員轉(zhuǎn)化生長因子(ransforming growth, TGF-β)之骨形態(tài)發(fā)生蛋白(bone morphogenetic protein)誘導(dǎo)骨髓問充質(zhì)干細(xì)胞的成骨分化,在目前的生物界中,一共有9個(gè)Smad家族成員。其中,Smad1、2、3、5、8為受體調(diào)節(jié)的Smad(receptor-regulated-Smad、R-Smad), Smad-4為共同的偶配體Smad(common-partner-Smad, Co-Smad-6、7為抑制性Smad(inhibitory Smad, I-Smad)。 BMPR-I使Smad-1、5、8末端絲氨酸殘基(SSXSmotif)磷酸化,隨后2個(gè)或1個(gè)R-Smad與1個(gè)Smad-4以異源三聚體或異源二聚體R-Smad-Co-Smad的形式進(jìn)入核內(nèi),作用于成骨細(xì)胞特異性轉(zhuǎn)錄因子Cbfa1、Osx等的基因序列上上調(diào)其表達(dá),從而增強(qiáng)成骨細(xì)胞ALP和OC的表達(dá)。從而促進(jìn)成骨細(xì)胞的分化和成熟。 Runt related gene2(Runx2)轉(zhuǎn)錄因子：Runx2,又稱Cbfal,或NMP2,或AML3,是Drosophila Runt蛋白的同源蛋白�，F(xiàn)已知,Runx2在骨骼的發(fā)育過程中參與間充質(zhì)細(xì)胞的凝聚,間充質(zhì)干細(xì)胞向成骨細(xì)胞的分化,軟骨細(xì)胞的肥大化,骨骼的血管侵入等多個(gè)步驟,如血管內(nèi)皮生長因子(vascular endothelia growth factor, VEGF)表達(dá)的組織特異性調(diào)節(jié)需要Runx2的參與,以促進(jìn)骨骼的血管侵入。成骨細(xì)胞和肥大軟骨細(xì)胞均表達(dá)Runx2,成骨細(xì)胞的分化和軟骨細(xì)胞的成熟均需要它的參與,Runx2是調(diào)節(jié)軟骨細(xì)胞最終成熟的關(guān)鍵因子。Runx2缺陷的小鼠實(shí)驗(yàn)證明,由于軟骨成骨和膜內(nèi)成骨均被排除,其骨骼結(jié)構(gòu)中完全沒有骨形成,小鼠在出生后不久死亡。Runx2突變是先天性顱鎖骨發(fā)育不良致病的遺傳學(xué)基礎(chǔ)。Runx2不發(fā)揮直接的成骨基因表達(dá)調(diào)節(jié)作用,它必須與多種蛋白相配合,在不同階段或細(xì)胞內(nèi)發(fā)揮不同作用,具有時(shí)空特征。所以,Runx2被稱為成骨的中樞調(diào)節(jié)因子。目前至少有12種Runx2異構(gòu)體被發(fā)現(xiàn),其中Runx2-Ⅰ作用于早期成骨細(xì)胞的分化和膜內(nèi)骨化過程,Runx2-Ⅱ作用與成骨細(xì)胞的成熟和軟骨內(nèi)骨化過程,其他異構(gòu)體的功能尚不清楚。 為此,本課題著重探討人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cells, HUVECs)在聯(lián)合培養(yǎng)體系中對人骨髓間充質(zhì)干細(xì)胞(human bone marrow Mesenchymal stem cells, hBMSCs)的形態(tài)、生長、細(xì)胞分化及其Smad-1基因和Runx2基因表達(dá)的影響,從基因水平驗(yàn)證血管內(nèi)皮細(xì)胞對骨髓間充質(zhì)干細(xì)胞成骨分化的作用。為臍靜脈內(nèi)皮細(xì)胞和骨髓間充質(zhì)干細(xì)胞聯(lián)合培養(yǎng)作為骨組織工程的種子細(xì)胞提供理論基礎(chǔ)。 [方法] (1)抽取志愿者骨髓液,使用密度梯度離心法分離骨髓單個(gè)核細(xì)胞,并借助hBMSCs黏附于塑料瓶底這一特性進(jìn)行純化,相差顯微鏡觀察形態(tài)變化。將hBMSCs傳代擴(kuò)增培養(yǎng)至第三代,流式細(xì)胞儀檢測CD34、CD29、CD44表面抗原表達(dá),鑒定hBMSCs; (2)將訂購的hUVECs用ECM+10%新生胎牛血清擴(kuò)增至第三代后與第三代hUVECs按1：1比例建立以DMEM+10%胎牛血清為培養(yǎng)基的hBMSCs和hUVECs聯(lián)合培養(yǎng)體系。以單獨(dú)培養(yǎng)的hBMSCs組及hUVECs組作為陰性對照組,分別于第4、6、8、10天相差顯微鏡觀察形態(tài)變化,用計(jì)數(shù)板計(jì)數(shù)各組hBMSCs數(shù)量； (3)分別于第4、6、8、10天每組每個(gè)時(shí)間點(diǎn)取6孔檢測三組培養(yǎng)體系中堿性磷酸酶(Alkaline phosphatase, ALP)及骨鈣素(Osteocalin, OC)含量。用SPSS17.0軟件對各項(xiàng)檢測值進(jìn)行統(tǒng)計(jì)學(xué)分析； (4)采用實(shí)時(shí)熒光定量PCR (real-time-PCR)檢測第4、6、8、10天單獨(dú)培養(yǎng)的hBMSCs組及聯(lián)合培養(yǎng)組中hBMSCs的Smad-1和基因Runx2表達(dá)的情況,每組每個(gè)時(shí)間點(diǎn)取6孔。用SPSS17.0軟件對各項(xiàng)檢測值進(jìn)行統(tǒng)計(jì)學(xué)分析。 [結(jié)果] (1)采用Ficoll液密度梯度離心法分離、提純hBMSCs可達(dá)到較高的純度。用流式細(xì)胞儀對第3代hBMSCs進(jìn)行細(xì)胞表型分析鑒定,CD34低表達(dá),CD29、CD44高表達(dá)；符合hBMSCs培養(yǎng)特征。 (2)新生胎牛血清分離培養(yǎng)的人骨髓問充質(zhì)干細(xì)胞成細(xì)長梭形,細(xì)胞為典型的成纖維細(xì)胞樣,呈漩渦狀貼壁生長,細(xì)胞較細(xì)小,倒置顯微鏡下觀察,可見接種后24h內(nèi)少部分單個(gè)核細(xì)胞貼壁,貼壁細(xì)胞成圓形,有小的胞質(zhì)突起。原代培養(yǎng)4至5天就可見成團(tuán)生長的細(xì)胞；第三代骨髓間充質(zhì)干細(xì)胞形態(tài)單一,成梭形,呈旋渦狀分布,有明顯的極性,沒有細(xì)胞重疊現(xiàn)象。hBMSCs在4-6天呈對數(shù)生長,8-10天后生長進(jìn)入平臺(tái)期,數(shù)量變化不太明顯。HUVECs約2-3天即可見細(xì)胞單層生長,形態(tài)呈多角形、鵝卵石狀鑲嵌排列,邊界清楚,胞漿豐富,胞核呈圓形或橢圓形,偶見雙核,第5d融合成片,第一代至第四代生長速度較快,2-3天即可傳代； (3)各組堿性磷酸酶檢測量隨時(shí)間延長先增高后降低,各時(shí)間聯(lián)合培養(yǎng)組ALP較高,8天時(shí)聯(lián)合培養(yǎng)組堿性磷酸酶最高；骨髓間充質(zhì)干細(xì)胞組和臍靜脈內(nèi)皮細(xì)胞組ALP基本沒有變化;聯(lián)合培養(yǎng)組和其它各組之間兩兩比較均有顯著統(tǒng)計(jì)學(xué)意義(P0.05)；各組骨鈣素檢測量隨時(shí)間延長先增高后降低；8天時(shí)聯(lián)合培養(yǎng)組骨鈣素最高；聯(lián)合培養(yǎng)組和其它各組之間兩兩比較均有顯著統(tǒng)計(jì)學(xué)意義(P0.05)； (4)聯(lián)合培養(yǎng)組Smad-1、Runx2基因檢測量隨時(shí)間延長逐漸增高,與其他組對比各時(shí)間聯(lián)合培養(yǎng)組Smad-1、Runx2基因表達(dá)均較高；第8天時(shí)聯(lián)合培養(yǎng)組Smad-1、Runx2基因檢測量最高；聯(lián)合培養(yǎng)組和骨髓間充質(zhì)干細(xì)胞組之間比較有顯著統(tǒng)計(jì)學(xué)意義(P0.05)。 [結(jié)論] (1)采用Ficoll液密度梯度離心法分離、提純的hBMSCs,經(jīng)流式細(xì)胞儀鑒定為較高純度的hBMSCs細(xì)胞； (2)骨髓間充質(zhì)干細(xì)胞與臍靜脈內(nèi)皮細(xì)胞聯(lián)合培養(yǎng)相容性良好,臍靜脈內(nèi)皮細(xì)胞對體外聯(lián)合培養(yǎng)體系中骨髓間充質(zhì)干細(xì)胞具有促進(jìn)增殖的作用; (3)在體外聯(lián)合培養(yǎng)體系中,臍靜脈內(nèi)皮細(xì)胞能促進(jìn)骨髓間充質(zhì)干細(xì)胞Smad1,Runx2因子的表達(dá),誘導(dǎo)其向成骨細(xì)胞方向分化。
[Abstract]:[background and purpose]
Various types of jaw defects are very common in clinic, but the traditional repair methods such as distraction osteogenesis, autologous bone transplantation, allograft bone transplantation are often constrained by the insufficient number of donors, donor lesions, secondary injury, potential antigenicity, infection and other factors and need to be improved urgently. The development of tissue engineering has brought hope to the repair of bone defect. Its purpose is to construct active tissue or organ by co-culture of functional cells and biodegradable three-dimensional scaffolds, and then implant them into the body to replace the damaged tissue and restore its morphology, structure and function. However, although tissue-engineered bone can form bone, there are some problems such as slow vascularization of tissue-engineered bone and unstable growth of new bone.
Human bone marrow mesenchymal stem cells (hBMSCs) have the potential to differentiate into adipocytes, osteoblasts, chondrocytes, cardiomyocytes, neurons, myoblasts, tendon cells and astrocytes under suitable conditions. Currently, the induction of simple hBMSCs into osteoblasts is discussed. Among them, BMP2, BMP4, BMP6 and BMP7 are the most representative ones, which can induce many kinds of cell proliferation, differentiation, apoptosis, participate in tissue regeneration and remodeling, and play a regulatory role in many kinds of life activities, such as cell migration and division. The bone cycle is long, the osteogenesis efficiency is low, and the cells are easy to be aged.
Endothelial cells can secrete bone morphogenetic protein (BMP), promote osteogenic differentiation and stimulate osteoblasts and their precursors to secrete vascular endothelial growth factor (VEGF), and vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and angiogenesis, and can promote endothelial fineness. However, the specific mechanism of vascular endothelial cells on osteogenic differentiation of bone marrow-derived mesenchymal stem cells is still unclear, and there is no gene level to verify the role of vascular endothelial cells on osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Bone morphogenetic protein (BMP), a member of the transforming growth factor family, induces osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). At present, there are nine Smad family members in the biological world. Among them, Smad1, 2, 3, 5, 8 are receptor-regulated Smad (R-Smad), Smad-4 are common. BMPR-I phosphorylates the Smad-1,5,8-terminal serine residue (SSXSmotif), and then two or one R-Smad and one Smad-4 enter the nucleus in the form of an allotrimer or a dimer, R-Smad-Co-Smad. Factor Cbfa1, Osx and other genes up-regulate their expression, thereby enhancing the expression of ALP and OC in osteoblasts, thereby promoting the differentiation and maturation of osteoblasts.
Runt related gene 2 (Runx2) transcription factor: Runx2, also known as Cbfal, or NMP2, or AML3, is a homologous protein of Drosophila Runt protein. Runx2 is known to participate in the process of bone development in the condensation of mesenchymal cells, mesenchymal stem cell differentiation into osteoblasts, chondrocyte hypertrophy, bone vascular invasion and other steps, such as blood. The tissue-specific regulation of vascular endothelial growth factor (VEGF) expression requires the involvement of Runx2 in promoting vascular invasion of bone. Both osteoblasts and mast chondrocytes express Runx2, which is involved in the differentiation of osteoblasts and the maturation of chondrocytes. Runx2 regulates the final maturation of chondrocytes. Runx2 mutation is the genetic basis for the pathogenesis of congenital skull and clavicle dysplasia. Runx2 does not play a direct role in the regulation of osteogenic gene expression. Runx2 is known as a central regulator of osteogenesis. At least 12 Runx2 isomers have been found, including Runx2-I, which acts on the differentiation and intramembranous ossification of early osteoblasts, Runx2-II, and the maturation and ossification of osteoblasts. The function of other isomers in the process of endochondral ossification is not clear.
In this study, we focused on the effects of human umbilical vein endothelial cells (HUVECs) on the morphology, growth, cell differentiation and the expression of Smad-1 and Runx2 genes of human bone marrow mesenchymal stem cells (hBMSCs) in a co-culture system. To investigate the effect of vascular endothelial cells on osteogenic differentiation of bone marrow mesenchymal stem cells and to provide theoretical basis for the co-culture of umbilical vein endothelial cells and bone marrow mesenchymal stem cells as seed cells of bone tissue engineering.
[method]
(1) Bone marrow mononuclear cells were isolated from the bone marrow of volunteers by density gradient centrifugation and purified by hBMSCs adherence to the bottom of plastic bottles. The morphological changes were observed by phase contrast microscope.
(2) The co-culture system of hBMSCs and hUVECs with DMEM+10% fetal bovine serum as medium was established by amplifying the ordered hUVECs with ECM+10% fetal bovine serum to the third generation and the third generation of hUVECs at a ratio of 1:1. The number of hBMSCs in each group was counted by counting board.
(3) Alkaline phosphatase (ALP) and osteocalcin (OC) were measured at 6 holes at each time point on the 4th, 6th, 8th and 10th day in each group.
(4) Real-time quantitative PCR (real-time-PCR) was used to detect the expression of Smad-1 and Runx2 in hBMSCs cultured on the 4th, 6th, 8th and 10th day respectively and in co-cultured groups. Six holes were taken from each group at each time point.
[results]
(1) High purity of hBMSCs was obtained by density gradient centrifugation with Ficoll solution. The phenotype of the third generation of hBMSCs was analyzed by flow cytometry. The results showed that the expression of CD34 was low, and that of CD29 and CD44 was high.
(2) Human bone marrow mesenchymal stem cells (BMSCs) isolated from fetal bovine serum (FBS) were spindle-shaped, and the cells were typical fibroblast-like cells, which grew in a whirlpool-like manner. The cells were smaller and smaller. Under inverted microscope, a small number of mononuclear cells adhered to the wall within 24 hours after inoculation. The adherent cells were round and had small cytoplasmic processes. The third generation of bone marrow mesenchymal stem cells showed single morphology, spindle-shaped, vortex-like distribution, obvious polarity, no cell overlap. hBMSCs grew logarithmically in 4-6 days, and entered plateau stage after 8-10 days, the number of cells did not change significantly. HUVECs could see cell monolayer growth in 2-3 days, morphology was polygonal. The nuclei were round or oval, occasionally binuclear, and fused into pieces on the 5th day. The growth rate of the first generation to the fourth generation was faster, and it could be subcultured in 2-3 days.
(3) Alkaline phosphatase (ALP) level in the combined culture group was higher than that in the combined culture group. ALP level in the combined culture group was the highest at 8 days. ALP level in the bone marrow mesenchymal stem cell group and umbilical vein endothelial cell group was almost unchanged. There was significant difference between the combined culture group and other groups (P Osteocalcin levels in each group increased first and then decreased with time prolonging, osteocalcin levels in the combined culture group were the highest at 8 days, and there was significant difference between the combined culture group and other groups (P 0.05).
(4) Smad-1, Runx2 gene detection increased gradually with time in the co-culture group, compared with other groups at all times in the co-culture group Smad-1, Runx2 gene expression were higher; on the eighth day in the co-culture group Smad-1, Runx2 gene detection was the highest; there was significant difference between the co-culture group and the bone marrow mesenchymal stem cell group (P 0.05). ).
[Conclusion]
(1) hBMSCs were purified by Ficoll density gradient centrifugation and identified as high purity hBMSCs by flow cytometry.
(2) Bone marrow mesenchymal stem cells and umbilical vein endothelial cells have good compatibility in co-culture. Umbilical vein endothelial cells can promote the proliferation of bone marrow mesenchymal stem cells in vitro co-culture system.
(3) Umbilical vein endothelial cells can promote the expression of Smad1 and Runx2 in bone marrow mesenchymal stem cells and induce them to differentiate into osteoblasts in vitro.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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